Whereas serum antibody (Ab) responses to HIV-1 envelope antigens are elicited in HIV-infected individuals, the initial Ab response is non-neutralizing and directed to epitopes that are poorly conserved among independent HIV-1 isolates (reviewed in Tomaras and Haynes, Curr. Opin. HIV AIDS 5:373-379 (2009)). Neutralizing Ab responses to HIV-1 do emerge in a significant minority of patients several months after infection (Shen et al, J. Virol. 83:3617-3625 (2009), Wei et al, Nature 422:307-312 (2003)) and select for resistant HIV-1 mutants (Burton et al, Nat. Immunol. 5:233 (2004)).
Among the conserved, neutralizing epitopes of HIV-1 is the membrane proximal external region (MPER) of gp41, a structure critical for viral fusion with target cell membranes (Wyatt and Sodroski, Science 280:1884-1888 (1998)). A series of neutralizing and broadly protective human Abs, 2F5, 4E10, and Z13, react with linear epitopes of the HIV-1 MPER (Muster et al, J. Virol. 67:6642-6647 (1993), Nelson et al, J. Virol. 81:4033-4043 (2007), Zwick et al, J. Virol. 75:10892-10905 (2001)) and yet are rarely elicited by infection (Shen et al, J. Virol. 83:3617-3625 (2009)). Indeed, despite significant effort, no vaccine or immunization strategy has been developed that that routinely induces robust MPER antibody responses (Coeffier et al, Vaccine 19:684-693 (2000), Derby et al, J. Virol. 80:8745-8762 (2006), Eckhart et al, J. Gen. Virol. 77(Pt9):2001-2008 (1996)).
A variety of hypotheses have been proposed to explain ineffective MPER Ab responses to HIV-1 infection and vaccines, including the complexity of HIV epitopes, high frequency of lentivirus mutation, shielding of crucial antigenic determinants by glycosylation, competitive suppression by non-neutralizing surface antigens, and insufficient diversity in the primary Ab repertoire (reviewed in Burton et al, Nat. Immunol. 5:233 (2004)). While each of these conjectures is plausible, the demonstration that the 2F5 and 4E10 MPER Abs avidly react with human and mouse self-antigens, including cardiolipin (Haynes et al, Science 308:1906-1908 (2005), Verkoczy et al, Proc. Natl. Acad. Sci. USA 107:181-186 (2010)), suggested an alternative explanation for the rarity of MPER Ab responses: if HIV-1 MPER neutralizing epitopes mimic host antigens, the normal processes of immunological tolerance could purge sets of MPER specific B cells and impair MPER Ab responses (Haynes et al, Hum. Antibodies 14:59-67 (2005)).
During their development, self-reactive, immature B cells are tolerized by apoptosis, receptor editing, or anergy (Erikson et al, Nature 349:331-334 (1991), Gay et al, J. Exp. Med. 177:999-1008 (1993), Hartley et al, Cell 72:325-335 (1993), Hartley et al, Nature 353:765-769 (1991), Nemazee and Burki, Nature 337:562-566 (1989), Tiegs et al, J. Exp. Med. 177:1009-1020 (1993)). Self-reactive B cells that are not purged in the bone marrow (BM) can remain susceptible to mechanisms of peripheral tolerance mechanisms that limit their capacity to respond to antigen ligands (Adams et al, Proc. Natl. Acad. Sci. USA 87:5687-5691 (1990)). Therefore, the fate of self-/HIV-reactive B cells should be investigated to determine whether: i) these cells are purged from the mature B-cell repertoire during their development in BM or ii) these cells are present in peripheral lymphoid tissues but held in an anergic state. This information is vital to the logical design of future vaccines that attempt to elicit Ab responses to the MPER of HIV.
The influence of tolerance on MPER-reactive B-cell development has recently been investigated by the generation of 2F5 VDJ “knock-in” (2F5 VDJ-KI) mice (Verkoczy et al, Proc. Natl. Acad. Sci. USA 107:181-186 (2010)). B-cell development in 2F5 VDJ-KI mice is blocked in the BM at the transition of small pre-B to immature B cells (Verkoczy et al, Proc. Natl. Acad. Sci. USA 107:181-186 (2010)). This developmental blockade is virtually identical to that observed in mice expressing B cell receptors (BCRs) for MHC (Nemazee and Burki, Nature 337:562-566 (1989)) or double-stranded DNA (Chen et al, Immunity 3:747-755 (1995)). Unlike the studies that utilize BCRs with known specificity to self-antigens (MHC and DNA), the self-antigen(s) that mediates the selection of 2F5 VDJ-KI B cell development has not yet been identified. Therefore, it is not clear whether this induction of tolerance in MPER-reactive B cells is mediated by interaction with cellular lipids or polypeptide antigens, as both 2F5 and 4E10 mAbs show significant but independent binding to each class of antigen (Alam et al, Proc. Natl. Acad. Sci. USA 105:20234-20239 (2009), Ofek et al, J. Virol. 84:2955-2962 (2010)).
Structural analyses of 2F5 and 4E10 mAbs indicate that their capacity to react with lipid antigens is potentiated by extended, hydrophobic HCDR3 motifs that have minimal interactions with the nominal MPER polypeptide (Alam et al, Proc. Natl. Acad. Sci. USA 105:20234-20239 (2009), Cardoso et al, Immunity 22:163-173 (2005), Ofek et al, J. Virol. 78:10724-10737 (2004)). Reciprocally, select mutations in the HCDR3 of 2F5 and 4E10 impair MPER polypeptide binding with little effect on lipid reactivity (Alam et al, Proc. Natl. Acad. Sci. USA 105:20234-20239 (2009)). Moreover, mutations of the 41-reactive and/or lipid-binding hydrophobic loop of HCDR3 regions will significantly reduce the ability of 2F5 and 4E10 to neutralize HIV infection (Alam et al, Proc. Natl. Acad. Sci. USA 105:20234-20239 (2009), Ofek et al, J. Virol. 84:2955-2962 (2010)), Scherer et al, Proc. Natl. Acad. Sci. USA 107:1529-1534 (2010)). These data suggest that removal of B cells that express Ab with either lipid-reactivity or the appropriate MPER peptide specificity would result in the loss of HIV neutralizing activity.
Without resorting to transgenic or “knock-in” mice, it is possible to identify specific B cells either by antigen-binding (Lalor et al, Eur. J. Immunol. 22:3001-3011 (1992), McHeyzer-Williams et al, J. Exp. Med. 178:295-307 (1993), McHeyzer-Williams et al, Nature 350:502-505 (1991)) or by anti-idotypic mAb (Reth et al, Eur. J. Immunol. 9:1004-1013 (1979), Takemori et al, Eur. J. Immunol. 12:1040-1046 (1982)). The development of B-cell tetramers, analogous to those routinely used to identify antigen-specific T cells (Altman et al, Science 274:94-96 (1996)), has greatly enhanced the ability to identify and isolate antigen-specific B cells despite their low frequencies (Newman et al, J. Immunol. Methods 272:177-187 (2003)). B-cell tetramers have been used to identify MPER peptide-reactive B cells within central and peripheral lymphoid tissues and to follow the fates of tetramer-binding cells in vivo. These B-cell tetramer reagents have been used to test the prediction that HIV gp41 MPER-reactive cells should be enriched in developmentally immature B-cell compartments but rare or absent in mature B-cell populations.
A BM culture system has been described that supports the survival, proliferation and differentiation of virtually all B2-lineage developmental stages (Holl et al, J. Immunol. Methods 354:53-67 (2010)). These culture-derived (CD) B-lineage cells are phenotypically and functionally similar to their in vivo counterparts (Holl et al, J. Immunol. Methods 354:53-67 (2010)) but develop in the absence of many self antigens and absent the environment of the BM (Sandel et al, J. Immunol. 166:5935-5944 (2001), Sandel and Monroe Immunity 10:289-299 (1999)). CD B cells are enriched for autoreactive specificities, and maintain this bias even after transfer to RAG1 deficient hosts (Holl et al, J. Immunol. Methods 354:53-67 (2010)). A determination has now been made as to whether B cells specific for the 2F5 peptide epitope of MPER, independent of association with lipids, are tolerized. Importantly, these cultures contain MPER-reactive B cells and mice reconstituted with CD B cells generated both robust germinal center (GC) responses and serum IgG Ab upon immunization with HIV peptide. In contrast, C57BL/6 animals did not respond to MPER immunization because these MPER-reactive B-cell subsets were lost beyond the BM transitional B cell stages in vivo.
Thus, the present invention results, at least in part, from studies demonstrating that MPER-reactive B cells are generated but are subsequently lost during T1 and T2 stages of B-cell development in the BM. The invention provides an adjuvant that breaks peripheral tolerance/anergy so that broadly neutralizing antibodies can be induced.